B. burgdorferi is maintained in nature in two very distinct environments, the Ixodes tick and mammalian hosts. Its ability to adapt and survive in these very different environments is attributed to its ability to sense changes in temperature, pH, cell density, oxygen and/or exposure to host factors and alter gene expression accordingly. The capability of bacteria to regulate transcription initiation allows for the coordinated expression of genes at appropriate times. Previous reports have demonstrated that central to the regulation of these responses are SigmaS (encoded by rpoS) and Sigma54 (encoded by rpoN also known as ntrA). In addition, Sigma54-dependent expression of SigmaS is responsible for the expression of key virulence factors [e.g., outer surface protein C (OspC), OspA and decorin-binding protein C (DbpC)] required for infectivity and transmission during the infective cycle. [unreadable] [unreadable] Promoters recognized by Sigma54-RNA polymerase holoenzyme (Sigma54-holoenzyme) have well conserved GG and GC doublets at Sigma4 and Sigma12 positions, respectively, relative to the transcriptional start site, instead of the typical -35/-10 boxes observed in Sigma70 class promoters. Universally, Sigma54-dependent transcription has been shown to require activator proteins, many of which are response regulators of two-component systems. In most cases, Sigma54-activator proteins bind enhancer-like elements located 100-200 bp upstream of the transcriptional start site of the promoter. Analysis of the genome of B. burgdorferi identified Rrp2 (BBO763) as a predicted Sigma54-dependent activator consisting of an N-terminal receiver domain, a central ATPase domain and a C-terminal DNA-binding domain.[unreadable] [unreadable] The activities of activators of Sigma54-holoenzyme are regulated in response to environmental signals. Many of the activators of #m54-holoenzyme are response regulators in two-component regulatory systems, and phosphorylation of these proteins results in their activation. These response regulators are phosphorylated by their cognate protein histidine kinases in response to an environmental signal. Once phosphorylated, the response regulator activates transcription of other genes. An activator of Sigma54-holoenzyme in B. burgdorferi, Rrp2 (encoded by rrp2), is also a response regulator of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, hk2 (encoded by hk2). The Sigma54-dependent activation of SigmaS requires response regulator, Rrp2 (BB0763). QRT-PCR experiments on reporter cat constructs in B. burgdorferi indicated that DNA sequences upstream of the Sigma54-dependent rpoS promoter were not needed by Rrp2 to activate transcription of rpoS. Further experiments showed that rpoN is required for stationary phase expression of rpoS in B. burgdorferi, but that the histidine kinase Hk2 is not absolutely essential. This suggested that another histidine kinase or small molecular weight phosphate donors could activate Rrp2, providing multiple signaling pathways for modulating expression of the Sigma54/SigmaS regulon in B. burgdorferi). We have also shown that rpoS has an additional Sogma54-independent promoter indicating that there are addition ways to regulate rpoS expression. The focus of this aspect of our research is to characterize a regulatory cascade involving SigmaS and Sigma54 in B. burgdorferi and to determine how this cascade is related to survival of the bacterium in the tick midgut and in a human host.